One major obstacle to the development of an effective HIV-1 vaccine is the difficulty in eliciting a broadly neutralizing antibody response. The envelope glycoprotein (Env) complex, the only HIV-1 target for neutralizing antibodies, exists in distinct conformational states on the cell or virion surface. Different oligomeric forms of Env are thought to reflect pleomorphic interactions of two heptad-repeat (HR) regions in the ectodomain of gp41. Intensive immunogen design efforts have been directed toward the creation of a soluble recombinant Env (gp140) protein that forms the mature trimeric structure found in functional spikes. This proposal grows directly out of our recent determination of the crystal structures of the two states of the C- terminal HR1 region of gp41: one, a trimeric coiled coil and the other, a tetrameric coiled coil. Our preliminary results favor the hypothesis that this structural dimorphism can inflence the stability and specificity of the oligomeric Env complex. We propose to use these insights to engineer and produce stabilized forms of the cleaved HIV-1 gp140 trimer as novel candidate immunogens for the induction of broadly neutralizing antibodies. The Specific Aims of the proposed research are: (1) To create homogeneous preparations of stabilized, cleaved HIV-1 gp140 trimers for immunogenicity studies. We will use mutational analysis to dissect the determinants of each of the two newly identified oligomeric coiled-coil interactions in the gp41 HR1 sequence motif. We will build on Moore's SOS method for making a trimeric, stabilized form of gp140 by selecting and testing the HR1 substitutions identified above that stabilize inter-subunit interactions in gp41. We will also make modifications of gp140 trimers in order to reduce pleomorphism and/or flexibility. (2) To evaluate the immunogenicity of stable, cleaved HIV-1 gp140 trimers in small animals and use the resulting data to improve immunogen design. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilization of the gp41-gp41 interactions affect the ability of HIV-1 gp140 trimers to elicit neutralizing antibodies. We will also evaluate the immunogenicity of trimeric gp140 complexes captured onto nanometer-sized beads as particulate immunogens. PUBLIC HEALTH RELEVANCE: The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control (1-4). Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require new and imaginative design strategies to bring us closer to the goal of a successful AIDS vaccine.